Dynamically-adaptive live therapeutic agents and methods of use thereof

ABSTRACT

This disclosure provides microbes engineered to detect virulent and spore states of pathogens and release an appropriate therapeutic response accordingly and compositions and methods of use of the same.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The United States Government has rights in this application pursuant toContract No. DE-AC52-07NA27344 between the United States Department ofEnergy and Lawrence Livermore National Security, LLC for the operationof Lawrence Livermore National Laboratory.

BACKGROUND

Current use, and the misuse or overuse, of broad spectrum antibioticshas significantly limited their effectiveness against pathogens,especially those pathogens that have life-cycles that can evade thesedrugs. As pathogens develop new resistance mechanisms, the ability totreat even common infectious diseases diminishes and results in highermedical expenses, prolonged illnesses, and even death.

Clostridium difficile (C. difficile or C. diff) bacterial infectionscause diarrhea as well as more serious intestinal conditions such ascolitis. C. difficile is a leading cause of intestinal infection and iscommon in people on prolonged antibiotic regimens, the elderly, andthose in hospitals. C. difficile has proven difficult to treat, withrecurrence resulting in more than 20% of patients. Recurrence is causedin part because C. difficile is naturally resistant to broad spectrumantibiotics due to formation of a dormant, spore state. Suppression ofnatural gut microbiota during antibiotic treatment further contributesto the spread and impact of C. difficile. In fact, C. difficile killsmore than 14,000 people a year in the United States alone and addsapproximately $4.8 billion in annual U.S. healthcare costs.

Novel approaches that can respond dynamically with the changing lifecycle states of resistant pathogens are needed.

SUMMARY

Methods and materials are provided for detecting and responding todifferent pathogen states.

In some aspects the disclosure provides genetically engineered microbescomprising a dual pathogen state detection system, the dual pathogendetection system comprising: (a) a first sensor for detection of avirulent form of a pathogen, wherein the first sensor is operativelyconnected to a virulent secretion system; and (b) a second sensor fordetection of a spore form of the pathogen, wherein the second sensor isoperatively connected to a spore secretion system.

In some aspects the disclosure provides compositions comprising anamount of the genetically engineered microbe according to any embodimentdisclosed and described herein.

In some aspects the disclosure provides methods of inhibiting apathogenic microbe comprising delivering a genetically engineeredmicrobe as in any embodiment disclosed and described herein to acomposition comprising the pathogenic microbe.

In some aspects the disclosure provides methods of detecting a state ofa pathogenic microbe comprising delivering the genetically engineeredmicrobe as in any embodiment disclosed and described herein to acomposition comprising the pathogenic microbe.

In some aspects the disclosure provides methods of treating orpreventing a pathogenic infection in a subject in need thereofcomprising administering to the subject an effective amount of acomposition comprising genetically engineered microbes as in anyembodiment disclosed and described herein.

In some embodiments, the microbe is a bacterium, for example, aGram-positive bacterium. In some embodiments, the bacterium isEscherichia coli Nissle 1917 or Lactococcus lactis (L. lactis). In someembodiments, the microbe is a viable microbe.

In some embodiments, at least the first sensor or the second sensordetects a protein on the surface of the pathogen.

In some embodiments, the modified cell surface receptor comprises anantibody, antimicrobial peptide, or fragment thereof.

In some embodiments, the first sensor detects a toxin secreted by thevirulent pathogen, for example, TcdA or TcdB.

In some embodiments, the second sensor detects a cell surface protein onthe pathogen.

In some embodiments, the cell surface protein is a cell wall protein ora spore coat protein, for example, a BclA glycoprotein or a cysteine(CdeC)-rich protein.

In some embodiments, at least one of the first sensor or the secondsensor does not detect a quorum sensing molecule.

In some embodiments, the virulent secretion system produces one or moreagents in an encapsulated shell, one or more Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) guide ribonucleic acids(gRNA), or any combination thereof.

In some embodiments, the encapsulated agent is a bacteriocin or amodified derivative thereof. In some embodiments, the bacteriocin isselected from the group consisting of colicin, diffocin, pyocin, andrhuricin 17.

In some embodiments, the encapsulated agent is an autolysin, anendolysin, an antimicrobial peptide, an antitoxin, or any combinationthereof.

In some embodiments, the encapsulated shell is modified with anantibody. In some embodiments, the antibody targets intestinalepithelial cells.

In some embodiments, the spore secretion system produces encapsulatedagents. In some embodiments, the encapsulated agents induce germinationof spores. In some embodiments, the encapsulated agents are bile salts.In some embodiments, the encapsulated agent is a protease.

In some embodiments, the microbe further comprises a microbe deathtrigger.

In some embodiments, the composition is a probiotic, a food, anutraceutical, a pharmaceutical, a biospray and/or a beverage. In someembodiments, the composition further comprises a pharmaceuticallyacceptable carrier/excipient.

In some embodiments, the pathogenic microbe is part of a population ofcultured cells (i.e., in vitro). In some embodiments, the pathogenicmicrobe is part of a population of cells of a subject (i.e., in vivo).

In some embodiments, the state is a virulent state. In otherembodiments, the state is a spore state.

In some embodiments, the pathogenic infection is a recurrent pathogenicinfection.

In some embodiments, the pathogen infection is caused by an intestinaland/or gastrointestinal pathogen, for example, Clostridium difficile.

In some embodiments, the subject experiences and/or reports fewer orless severe side effects on natural gut microbiota as compared to aconventional therapy, for example, the conventional therapy is anantibiotic.

In some embodiments, the genetically engineered microbes are locallyadministered.

In some embodiments, the composition comprising genetically engineeredmicrobes is administered sequentially or concurrently with anantibiotic. In some embodiments, the composition is administered afterthe antibiotic.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a schematic of the screening process of cell bindingdomains/antibodies, endolysins and antimicrobial peptides for developingengineered cell surface signaling receptors that have virulent bindingdomains and that initiate release of a CRISPR/phage therapy that targetand kill pathogens.

FIG. 2 presents several schematics of modular pathogen state detectionsystems according to an embodiment of the present disclosure.

FIG. 3 presents a schematic of a therapeutic microbe according to anembodiment of the present disclosure, engineered to detect and respondto both virulent and spore forms of a pathogen and to calculate when andwhether to undergo lysis to release a therapeutic agent.

FIG. 4 presents a schematic of a therapeutic agent according to anembodiment of the present disclosure, engineered to sense the changingstate of a pathogen within a host, process the information usingengineered signaling cascades, and deliver specific, scalable, andeffective countermeasures against the sensed state. Upon detection of aspore coat protein on the surface of the dormant spore form, theengineered microbe releases a spore-targeting agent. Upon binding of atoxin (circles) released from the virulent form, the engineered microbereleases via lysis an encapsulated virulent targeting agent.

FIG. 5 presents a general schematic of a dynamically adaptivetherapeutic agent according to an embodiment of the present disclosure,engineered to be able to detect and respond to both spore and virulentforms of C. difficile, as well as mitigate toxic effect of toxin on hosttissues.

DETAILED DESCRIPTION

Conventional treatments for pathogenic infections often utilize broadspectrum antibiotics. Such antibiotics, like ampicillin for example,revolutionized medicine for their ability to act against a wide range ofdisease causing bacteria. However, these antibiotics indiscriminatelytarget both the pathological bacteria and the natural, beneficialmicrobiota. Destruction of the body's natural bacteria provides anenvironment for drug resistant microbes to prosper and lead to secondaryinfection. One common secondary infection is C. diff. Importantly, useof “silver bullet” antibiotics as a single treatment regimen often doesnot meet all the requirements for efficacy, drug delivery, and low hosttoxicity. In particular, pathogens adapt and evolve to these statictreatments such that the treatments become ineffective.

The present disclosure relates to the use of live therapeutic agents(i.e., genetically engineered microbes) that can dynamically sense andrespond to the current state of a pathogen. Using multiple mechanisms ofattack, these agents act, in part, to slow down the evolution ofresistance. These therapeutic agents can be specifically tailored toidentify and respond to a particular pathogen and can be locallyreleased to minimize the effects on the host's tissue and naturalmicrobiota.

After reading this description it will become apparent to one skilled inthe art how to implement the invention in various alternativeembodiments and alternative applications. However, all the variousembodiments of the present invention will not be described herein. Itwill be understood that the embodiments presented here are presented byway of example only, and not limitation. As such, this detaileddescription of various alternative embodiments should not be construedto limit the scope or breadth of the present invention as set forthbelow.

The detailed description is divided into various sections only for thereader's convenience and disclosure found in any section may be combinedwith that in another section. Titles or subtitles may be used in thespecification for the convenience of a reader, which are not intended toinfluence the scope of the present disclosure.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, cell biology and recombinant DNA, which are withinthe skill of the art. See, e.g., Sambrook and Russell eds. (2001)Molecular Cloning: A Laboratory Manual, 3^(rd) edition; the seriesAusubel et al. eds. (2007) Current Protocols in Molecular Biology; theseries Methods in Enzymology (Academic Press, Inc., N.Y.); McPherson etal. (1991) PCR 1: A Practical Approach (IRL Press at Oxford UniversityPress); McPherson et al. (1995) PCR 2: A Practical Approach; Harlow andLane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005)Culture of Animal Cells: A Manual of Basic Technique, 5^(th) edition;Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195;Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson(1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984)Transcription and Translation; IRL Press (1986) Immobilized Cells andEnzymes; Perbal (1984) A Practical Guide to Molecular Cloning; Millerand Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (ColdSpring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); Herzenberg et al. eds (1996) Weir's Handbook of ExperimentalImmunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3^(rd)edition (2002) Cold Spring Harbor Laboratory Press; Sohail (2004) GeneSilencing by RNA Interference: Technology and Application (CRC Press).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the disclosure also contemplates that in someembodiments any feature or combination of features set forth herein canbe excluded or omitted. To illustrate, if the specification states thata complex comprises components A, B and C, it is specifically intendedthat any of A, B or C, or a combination thereof, can be omitted anddisclaimed singularly or in any combination.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate, oralternatively by a variation of +/−15%, or alternatively 10%, oralternatively 5%, or alternatively 2%. It is to be understood, althoughnot always explicitly stated, that all numerical designations arepreceded by the term “about.” It is to be understood that such rangeformat is used for convenience and brevity and should be understoodflexibly to include numerical values explicitly specified as limits of arange, but also to include all individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly specified. For example, a ratio in the range of about 1 toabout 200 should be understood to include the explicitly recited limitsof about 1 and about 200, but also to include individual ratios such asabout 2, about 3, and about 4, and sub-ranges such as about 10 to about50, about 20 to about 100, and so forth. It also is to be understood,although not always explicitly stated, that the reagents describedherein are merely exemplary and that equivalents of such are known inthe art.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of cells.

Definitions

As used herein the following terms have the following meanings:

The term “about,” as used herein when referring to a measurable valuesuch as an amount or concentration and the like, is meant to encompassvariations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specifiedamount.

The terms or “acceptable,” “effective,” or “sufficient” when used todescribe the selection of any components, ranges, dose forms, etc.disclosed herein intend that said component, range, dose form, etc. issuitable for the disclosed purpose.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

“Comprising” or “comprises” is intended to mean that the compositionsand methods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination for the stated purpose. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

As used herein the term “operatively connected,” such as in reference toa sensor for detection of a pathogen and a secretion system means thesensor and the secretion system are connected in such a way that theywork together, for example, via cell signaling. The elements that areoperatively connected do not need to actually touch, but one elementacts on the other.

Microbes

Aspects of the disclosure provide genetically engineered microbes.Suitable microbes include, for example, bacteria (e.g., Lactobacillus),yeast (e.g., Saccharomyces and Candida), and algal. In some embodiments,the microbe is a bacterium, for example a Gram-positive or aGram-negative bacterium.

Non-limiting examples of suitable bacteria include Acetobacter spp.,Acidithiobacillus spp., Aeromonas spp., Agrobacterium spp., Alcaligenesspp., Arthrobacter spp., Azotobacter spp., Bacillus spp.,Chromobacterium spp., Citrobacter spp., Clostridium spp., Comamonasspp., Corynebacterium spp., Escherichia spp., Flavobacterium spp.,Geobacillus spp., Geobacter spp., Gluconobacter spp., Lactobacillusspp., Lactococcus spp., Microlunatus spp., Mycobacterium spp., Pantoeaspp., Pseudomonas spp., Ralstonia spp., Rhizobium spp., Rhodococcusspp., Saccharopolyspora spp., Salmonella spp., Serratia spp.,Sinorhizobium spp., Stenotrophomonas spp., Streptococcus spp.,Streptomyces spp., Synechocystis spp., Thermus spp., Xanthomonas spp.,and Zymonas spp.

In some embodiments, the bacterium is a probiotic. Probiotics are livemicroorganisms, which when administered in adequate amounts confer ahealth benefit on the host. Non-limiting examples of probiotic bacteriainclude Escherichia coli Nissle 1917, Lactococcus Lactis, LactobacillusAcidophilus, Lactobacillus Brevis, Lactobacillus Bulgaricus,Lactobacillus Casei, Lactobacillus Helveticus, Lactobacillus reuteri,Lactobacillus rhamnosus, Lactobacillus Rhamnosus GG, Lactobacillusrhamnosus GR-1, Lactobacillus plantarum, Lactobacillus Silivarius,Eubacterium hallii and Bifidobacterum Bifidum, Bifidobacterum Breve,Bifidobacterium Infantis, Bifidobacterium Lactis, Bifidobacteriumlongum, Bacillus Coagulans, Saccharomyces Boulardii, StreptococcusThermophilus or a combination thereof. In one preferred embodiment, thebacteria is Lactococcus Lactis (L. lactis).

Non-limiting examples of suitable yeast include Brettanomyces spp.,Candida spp., Debaryomyces spp., Kluyveromyces spp., Pachysolen spp.,Paffia spp., Pichia spp., Saccharomyces spp., Schizosaccharmoyces spp.,Talaromyces spp., and Yarrowia spp. In one embodiment, the yeast is amodified Saccharmoyces cerevisiae.

In some embodiments, the microbe is a viable microbe.

Engineered Microbes

Aspects of the disclosure encompass genetically engineered microbecomprising a pathogen state detection system, in particular a dualpathogen state detection system. The detection systems comprise a sensoron the surface of the microbe that detects the presence of a pathogen.Detection can occur directly (i.e., detection of a pathogen cell surfacemarker) or indirectly (i.e., detection of a signal produced by thepathogen (e.g., a toxin). In a preferred embodiment, the dual pathogenstate detection system of the microbes comprise at least a first sensorfor detection of a virulent form of a pathogen and a second sensor fordetection of a spore form of the pathogen. The terms “state” and “form”can be used interchangeably in reference to a pathogen. For example, thespore state is equivalent to the spore form.

In some embodiments, at least the first sensor or the second sensordetects a protein on the surface of the pathogen. In other embodiments,the first sensor detects a protein on the surface of the pathogen andthe second sensor detects a non-surface pathogen signal. In yet otherembodiments, the second sensor detects a protein on the surface of thepathogen and the first sensor detects a non-surface pathogen signal. Instill other embodiments, both the first sensor and the second sensordetect a protein on the surface of the pathogen.

In some embodiments, the first sensor, the second sensor, or both is amodified cell surface receptor. A cell surface receptor can be modifiedby any method known in the art. Non-limiting examples of modified cellsurface receptors include antibodies, endolysins, and antimicrobialpeptides, and fragments thereof. In some embodiments, the modified cellsurface receptor comprises an antibody, antimicrobial peptide, orfragment thereof.

In one embodiment, the antibody or fragment thereof is a monoclonalantibody. In another embodiment, the antibody is a polyclonal. Suitableantibodies or fragments thereof for use with the present disclosureinclude, for example, actoxumab, bezlotoxumab, anthem, pagibaximab,efibazumab, urtoxazumab. As used herein the term “antibody” or“antibodies” refers to immunoglobulin molecules and immunologicallyactive portions or immunoglobulin molecules (i.e., molecules thatcontain an antigen binding site that immune-specifically bind anantigen). The term also refers to antibodies comprise of twoimmunoglobulin heavy chains and two immunoglobulin light chains as wellas any variety of forms including full length antibodies and portionsthereof; including, for example, an immunoglobulin molecule, amonoclonal antibody, a chimeric antibody, a CDR-grafted antibody, ahumanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linkedFv, a scFV, a single domain antibody (dAb), a diabody, a multispecificantibody, a dual specific antibody, an anti-idiotypic antibody, abispecific antibody, a functionally active epitope-binding fragmentthereof, bifunctional hybrid antibodies (e.g., Lanzavechhia et al.,(1987) Eur. J Immunol. 17, 105) and single chains (e.g., Huston et al.,Proc. Natl. Acad Sci. U.S.A., (1988) 85, 5879-5883 and Bird et al.,(1988) Science 242, 423-426, which are incorporated herein byreference). (See, generally, Hood et al., Immunology, Benjamin, N.Y.,2ND ed. (1984); Harlow and Lane, Antibodies. A Laboratory Manual, ColdSpring Harbor Laboratory (1988); Hunkapiller and Hood, (1986) Nature,323, 15-16, which are incorporated herein by reference). The antibodymay be of any type (e.g., IgG, IgA, IgM, IgE or IgD). Preferably, theantibody is IgG. An antibody may be non-human (e.g., from mouse, goat,or any other animal), fully human, humanized, or chimeric.

Antimicrobial peptides (AMPs) are a component of the innate immunesystem and play a critical role in warding off invading pathogens. Todate, more than 2,500 peptides have been isolated and characterized.Wang et al, (2004) Nucleic Acids Res. 32(Database issue): D590-2.Suitable AMPS include, for example, HBD1-3, HNP1, HD5 cathelicidins(e.g., LL-37), coprisin, polymixin, and nisin.

In one preferred embodiment, at least one of the first sensor or thesecond sensor does not detect a quorum sensing molecule. A quorumsensing molecule refers to a molecule produced by a cell that can signalpopulation density of the population of cells containing that cell. Insome embodiments, a quorum sensing molecule is an oligopeptide, anN-Acyl homoserine lactone (AHL), an autoinducer or a pheromone.

Virulent Detection and Secretion System

The disclosure provides a first sensor for detection of a virulent formof a pathogen, wherein the first sensor is operatively connected to avirulent secretion system.

In some embodiments, the first sensor detects a compound, for example atoxin, secreted from the pathogen. Bacterial generate toxins which areclassified as exotoxins or endotoxins. Exotoxins are secreted, whileendotoxins remain part of the bacteria. In some embodiments, the firstsensor detects large clostridial toxins (LCTs) produced by Clostridiumdifficile, Clostridium sordellii, Clostridium perfringens, orClostridium novyi. In some embodiments, the toxin is C. difficile toxinA (TcdA), toxin B (TcdB), or binary toxin A (CDTa). Toxin A is anenterotoxin that causes diarrhea. Toxin B is a cytotoxin that killscells.

Other non-limiting examples of bacterial toxins include, Bordetellapertussis AC toxin (A/B) and Bacillus anthracis EF, Botulinum neurotoxin(BoNT), tetanus toxin (TeNT protein), staphylococcal toxin, alpha toxin,anthrax toxin, cholera toxin, cyanotoxin, diphtheria toxin, E. coliheat-labile toxin LT, pertussis toxin, Pseudomonas toxin A, shiga toxin,shiga-like toxin, and Staphylococcus aureus Exfoliatin B.

In some embodiments, the first sensor detects a cell surface protein onthe surface of the virulent pathogen. In some embodiments, the cellsurface protein is Cwp66 adhesin protein, S-layer proteins (e.g.,HMW-SLP), or flagellin protein (e.g., FlaA, FlgE). In some embodiments,the cell surface protein is a spore coat proteins, for example, BclAglycoproteins or CdeC.

In some embodiments, the first sensor detects a catalytic or cell wallbinding domain of C. difficile. In some embodiments, the catalytic orcell wall binding domain from C. difficile includes any one of SEQ IDNOS. 1-36 or a portion thereof.

In some embodiments, the first sensor detects a catalytic or cell wallbinding domain of Clostridium phages. In some embodiments, the catalyticor cell wall binding domain from Clostridium phages includes any one ofSEQ ID NOS. 37-40 or a portion thereof.

In one preferred embodiment, first sensor for detection of a virulentform of a pathogen does not detect a quorum sensing molecule.

The microbes of the present disclosure also provide a virulent secretionsystem which is operatively connected to the first sensor.

The virulent secretion system can produce one or more agents in anencapsulated shell, one or more Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) guide ribonucleic acids (gRNA), or anycombination thereof. An example of CRISPR targets include Frizzledproteins (as described in Tao et al. (2016) Nature 538:350-355. In someembodiments, the CRISPR target is a toxin gene, for example, TcdA orTcdB. In some embodiments, the encapsulated agent is a bacteriocin, suchas, colicin, diffocin, pyocin, and rhuricin 17, or a modified derivativethereof. In other embodiments, the encapsulated agent is an autolysin,an endolysin, an antimicrobial peptide, an antitoxin, or any combinationthereof.

The encapsulation shells can be created by any method known in thefield, for example, by the method described by Moon et al. (2014) whichis hereby incorporated by reference in its entirety. Moon et al. (2014)Biomacromolecules 15 (10), 3794-3801. The encapsulation shells can bemodified by any known method. For example, the encapsulated shell ismodified with an antibody. In one embodiment, the antibody targetsintestinal epithelial cells.

Spore Detection and Secretion System

The disclosure also provides a second sensor for detection of a sporeform of the pathogen, wherein the second sensor is operatively connectedto a spore secretion system.

In some embodiments, the second sensor detects a cell surface protein onthe spore pathogen, for example, a cell wall protein or a spore coatprotein. Non-limiting examples of spore coat proteins include a BclAglycoprotein and a cysteine (CdeC)-rich protein.

In one preferred embodiment, the second sensor for detection of a sporeform of a pathogen does not detect a quorum sensing molecule.

The microbes of the present disclosure also provide a spore secretionsystem which is operatively connected to the second sensor. In someembodiments, the spore secretion system produces encapsulated agents.

In some embodiments, wherein the encapsulated agents induce germinationof spores, for example, bile salts. In other embodiments, theencapsulated agent is a protease.

In some embodiments, the microbes disclosed herein further comprise amicrobe death trigger (e.g., a suicide cassette). Cells may containsuicide cassettes comprising inserted sequences encoding certainreporter proteins (e.g., thymidine kinase (TK)). In some embodiments,suicide cassettes are used to facilitate the identification of cellsfrom a larger cell population. In other embodiments, suicide cassettesare used to destroy microbes which have proliferated to an undesirablelevel in vivo. Microbes can be further modified to comprise a trackingsystem to follow the in vivo position and ultimately final locationand/or clearance of the microbes following introduction into a subject.For example, the engineered microbes may be modified to comprise afluorescent detectable marker (e.g., green fluorescent protein (GFP)).

The microbes of the present disclosure can be genetically modified byany suitable methodology. As a non-limiting example, one or more of thenucleic acids (e.g., nucleic acid encoding for the first sensor, thesecond sensor, the virulent secretion system, and/or the spore secretionsystem) associated with the disclosure can be expressed in a recombinantexpression vector. As used herein, a “vector” may be any of a number ofnucleic acids into which a desired sequence or sequences may beinserted, such as by restriction and ligation, for transport betweendifferent genetic environments or for expression in a host cell. Vectorsare typically composed of DNA, although RNA vectors are also available.Vectors include, but are not limited to: plasmids, fosmids, phagemids,virus genomes and artificial chromosomes.

A cloning vector is one which is able to replicate autonomously orintegrated in the genome in a host cell, and which can be furthercharacterized by one or more endonuclease restriction sites at which thevector may be cut in a determinable fashion and into which a desired DNAsequence may be ligated such that the new recombinant vector retains itsability to replicate in the host cell. In the case of plasmids,replication of the desired sequence may occur many times as the plasmidincreases in copy number within the host cell such as a host bacteriumor just a single time per host before the host reproduces by mitosis. Inthe case of phage, replication may occur actively during a lytic phaseor passively during a lysogenic phase.

An expression vector is one into which a desired nucleic acid sequencemay be inserted, for example by restriction and ligation, such that itis operably joined to regulatory sequences and may be expressed as anRNA transcript. Vectors may further contain one or more marker sequencessuitable for use in the identification of cells which have or have notbeen transformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase, luciferase or alkaline phosphatase),and genes which visibly affect the phenotype of transformed ortransfected cells, hosts, colonies or plaques (e.g., green fluorescentprotein). Preferred vectors are those capable of autonomous replicationand expression of the structural gene products present in the DNAsegments to which they are operably joined. When the nucleic acidmolecule that encodes any of the genes associated with the claimedinvention is expressed in a cell, a variety of transcription controlsequences (e.g., promoter/enhancer sequences) can be used to direct itsexpression. The promoter can be a native promoter, i.e., the promoter ofthe gene in its endogenous context, which provides normal regulation ofexpression of the gene. In some embodiments the promoter can beconstitutive, i.e., the promoter is unregulated allowing for continualtranscription of its associated gene. A variety of conditional promotersalso can be used, such as promoters controlled by the presence orabsence of a molecule.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, FourthEdition, Cold Spring Harbor Laboratory Press, 2012. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA). That heterologous DNA (RNA) is placed underoperable control of transcriptional elements to permit the expression ofthe heterologous DNA in the host cell. A nucleic acid molecule thatcomprises a gene associated with the invention can be introduced into acell or cells using methods and techniques that are standard in the art.

A nucleic acid, polypeptide or fragment thereof described herein can besynthetic. As used herein, the term “synthetic” means artificiallyprepared. A synthetic nucleic acid or polypeptide is a nucleic acid orpolypeptide that is synthesized and is not a naturally produced nucleicacid or polypeptide molecule (e.g., not produced in an animal ororganism). It will be understood that the sequence of a natural nucleicacid or polypeptide (e.g., an endogenous nucleic acid or polypeptide)may be identical to the sequence of a synthetic nucleic acid orpolypeptide, but the latter will have been prepared using at least onesynthetic step.

Compositions

The disclosure also provides compositions comprising an amount of thegenetically engineered microbes disclosed and described herein. In someembodiments, the composition is a probiotic, a food, a nutraceutical, apharmaceutical, a biospray and/or a beverage.

In some embodiments, the composition further comprises apharmaceutically acceptable carrier/excipient. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The composition may comprise a pharmaceutically acceptable excipient, apharmaceutically acceptable salt, diluents, carriers, vehicles and suchother inactive agents well known to the skilled artisan. Vehicles andexcipients commonly employed in pharmaceutical preparations include, forexample, talc, gum Arabic, lactose, starch, magnesium stearate, cocoabutter, aqueous or non-aqueous solvents, oils, paraffin derivatives,glycols, etc. Solutions can be prepared using water or physiologicallycompatible organic solvents such as ethanol, 1,2-propylene glycol,polyglycols, dimethylsulfoxide, fatty alcohols, triglycerides, partialesters of glycerine and the like. Compositions may be prepared usingconventional techniques that may include sterile isotonic saline, water,1,3-butanediol, ethanol, 1,2-propylene glycol, polyglycols mixed withwater, Ringer's solution, etc. In one aspect, a coloring agent is addedto facilitate in locating and properly placing the composition to theintended treatment site.

Compositions may include a preservative and/or a stabilizer.Non-limiting examples of preservatives include methyl-, ethyl-,propyl-parabens, sodium benzoate, benzoic acid, sorbic acid, potassiumsorbate, propionic acid, benzalkonium chloride, benzyl alcohol,thimerosal, phenylmercurate salts, chlorhexidine, phenol, 3-cresol,quaternary ammonium compounds (QACs), chlorbutanol, 2-ethoxyethanol, andimidurea.

To control tonicity, the composition can comprise a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride and calcium chloride.

Compositions may include one or more buffers. Typical buffers include: aphosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, ahistidine buffer, or a citrate buffer. Buffers will typically beincluded at a concentration in the 5-20 mM range. The pH of acomposition will generally be between 5 and 8, and more typicallybetween 6 and 8 e.g. between 6.5 and 7.5, or between 7.0 and 7.8.

In some embodiments, the composition may include a cryoprotectant agent.Non-limiting examples of cryoprotectant agents include a glycol (e.g.,ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide(DMSO), formamide, sucrose, trehalose, dextrose, and any combinationsthereof.

The genetically engineered microbes may be formulated in a compositionfor oral administration, for example, as a tablet, capsule, or drink.The genetically engineered microbes may be combined in a liquid carrier.Preferably, the liquid carrier is a moving fluid and the term “liquidcarrier” refers to any liquid suitable for ingestion and includespharmaceutical formulations and foodstuffs such as water, milk, fruitjuices, vegetable juices, electrolytic beverages and the like.

The composition can comprise one or more additional substances that canbe consumed by the genetically engineered microbe to keep the relevantmicrobe alive or stimulate its growth. Non-limiting examples ofadditional substances include mucopolysaccharides, oligosaccharides,polysaccharides, amino acids, vitamins, nutrient precursors and proteins

The composition can be included in an implantable device. Suitableimplantable devices contemplated by this invention includeencapsulation, scaffolds, grafts, and the like. Such implantable devicescan be coated on at least one surface, impregnated, or encapsulated witha composition disclosed and described herein.

Methods of Use

Also provided are methods for killing or inhibiting a pathogenic microbecomprising delivering the genetically engineered microbe as disclosedand described herein to a composition comprising the pathogenic microbe.In some embodiments, the pathogenic microbe is a bacterial pathogen, aviral pathogen, or a fungal pathogen.

In some embodiments, the pathogenic microbe is part of a population ofcultured cells (i.e., in vitro). In other embodiments, the pathogenicmicrobe is part of a population of cells of a subject (i.e., in vivo).

Also provided herein are methods of detecting a state (e.g., virulent orspore) of a pathogenic microbe comprising delivering the geneticallyengineered microbe as disclosed and described herein to a compositioncomprising the pathogenic microbe. In some embodiments, the pathogenicmicrobe is part of a population of cultured cells (i.e., in vitro). Inother embodiments, the pathogenic microbe is part of a population ofcells of a subject (i.e., in vivo).

Also provided herein are methods of treating or preventing a pathogenicinfection in a subject in need thereof comprising administering to thesubject an effective amount of a composition comprising geneticallyengineered microbes as disclosed and described herein.

Subjects treated by the methods disclosed herein include, a simian, abovine, an equine, a canine, a murine, or a human patient.

In some embodiments, the pathogenic infection is a recurrent pathogenicinfection.

In one embodiment, the compositions described herein are useful intreating bacterial infections. Infectious bacteria include, withoutlimitation, Bacillus spp.; Bordetella spp.; Borrelia spp.; Brucellaspp.; Burkholderia spp.; Campylobacter spp.; Chlamydia spp.;Chlamydophila spp.; Clostridium spp.; Corynebacterium spp.; Enterococcusspp.; Escherichia spp.; Francisella spp.; Haemophilus spp.; Helicobacterspp.; Legionella spp.; Leptospira spp.; Listeria spp.; Mycobacteriumspp.; Mycoplasma spp.; Neisseria spp.; Pseudomonas spp.; Rickettsiaspp.; Salmonella spp.; Shigella spp.; Staphylococcus spp.; Streptococcusspp.; Treponema spp.; Vibrio spp.; and Yersinia spp. In one embodiment,the bacterial pathogen is a Clostridium difficile (C. diff). In someembodiments, the bacterial pathogen is an antibiotic-resistant bacterialpathogen. In some embodiments, the bacterial pathogen is a hypervirulentstrain.

Non-limiting examples of diseases caused by bacterial pathogens includeacute enteritis, anthrax, bacterial meningitis, botulism, brucellosis,cholera, community-acquired respiratory infection, diptheria, dysentery,hemolytic-uremic syndrome, hemorrhagic colitis, leprosy, lyme disease,lymphogranuloma venereum, neumonia, nongonococcal urethritis, sepsis,syphilis (e.g., congential syphilis), tetanus, tuberculosis, typhoidfever, whooping cough, trachoma, inclusion conjunctivitis of thenewborn, psittacosis, pseudomembranous colitis, gas gangrene, foodpoisoning, anaerobic cellulites, nosocomial infections, urinary tractinfections, diarrhea, tularemia, upper respiratory tract infections,bronchitis, peptic ulcers, legionnaire's disease, pontiac fever,leptospirosis, listeriosis, tuberculosis, gonorrhea, ophthalmianeonatorum, septic arthritis, meningococcal disease,Waterhouse-Friderichsen syndrome, Pseudomonas infection, rocky mountainspotted fever, typhoid fever type salmonellosis (dysentery, colitis),Salmonellosis with gastroenteritis and/or enterocolitis, bacillarydysentery/Shigellosis, coagulase-positive staphylococcal infections(such as impetigo, acute infective endocarditis, septicemia, necrotizingpneumonia, and toxinoses such as toxic shock syndrome or Staphylococcalfood poisoning), cystitis, septicemia, endometritis, otitis media,sinusitis, Streptococcal pharyngitis, scarlet fever, rheumatic fever,erysipelas, puerperal fever, necrotizing fascilitis, bubonic plague andpneumonic plague.

In some embodiments, the pathogen infection is caused by an intestinaland/or gastrointestinal pathogen. Gastrointestinal pathogens includepathogens (e.g., bacteria) that can colonize in the gut of a subject andcause and/or do cause a disease or condition in the subject. Exemplarygastrointestinal pathogens include, but are not limited to Escherichiacoli, Clostridium difficile, Clostridium perfringens, Listeriamonocytogenes, Listeria innocua, Staphylococcus aureus, Enterococcusfaecalis (virulent strains of E. faecalis), and Enterococcus faecium.

In one embodiment, the compositions described herein are useful intreating infections by pathogenic viruses. Pathogenic viruses include,without limitation, human papillomavirus, human immunodeficiency virus,Epstein-Barr virus, cytomegalovirus, Ebola virus, Marburg virus,influenza, respiratory syncytial virus, poxvirus, varicella-zostervirus, and herpes.

Non-limiting examples of viral pathogens include viruses belonging tothe following families: Adenoviridae (e.g., adenovirus), Picornaviridae(e.g., coxsackievirus, hepatitis A virus, poliovirus and rhinovirus),Herpesviridae (e.g., herpes simplex type 1, herpes simplex type 2,Varicella-zoster virus, Epstein-barr virus, human cytomegalovirus, andhuman herpesvirus type 8), Hepadnaviridae (e.g., hepatitis B virus),Flaviviridae (e.g., hepatitis C virus, yellow fever virus, dengue virus,and West Nile virus), Retroviridae (e.g., human immunodeficiency virus(HIV)), Orthomyxoviridae (e.g., influenza virus), Paramyxoviridae (e.g.,measles virus, mumps virus, parainfluenza virus, respiratory syncytialvirus and human metapneumovirus), Papillomaviridae (e.g.,papillomavirus), Rhabdoviridae (e.g., rabies virus), Togaviridae (e.g.,ubella virus) and Parvoviridae (e.g., human bocavirus and parvovirusB19).

Non-limiting examples of diseases caused by viral pathogens include:acute febrile pharyngitis, pharyngoconjunctival fever, epidemickeratoconjunctivitis, infantile gastroenteritis, Coxsackie infections,infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronichepatitis, hepatic cirrhosis, hepatocellular carcinoma, primary HSV-1infection, gingivostomatitis, tonsillitis, pharyngitis, primary HSV-2infection, latent HSV-2 infection, aseptic meningitis, infectiousmononucleosis, cytomegalic inclusion disease, Kaposi's sarcoma,Castleman disease, primary effusion lymphoma, AIDS, influenza, Reyesyndrome, measles, postinfectious encephalomyelitis, mumps, hyperplasticepithelial lesions, laryngeal papillomas, epidermodysplasiaverruciformis, croup, pneumonia, bronchiolitis, common cold, rabies,German measles, congenital rubella, varicella and herpes zoster.

The microbial compositions described herein are useful in treatinginfections caused by other microbes, including fungus and yeast

Cells can be administered to such an individual by absolute numbers ofcells, e.g., said individual can be administered from about 1000cells/injection to up to about 10 billion cells/injection, such as atabout, at least about, or at most about, 1×10⁸, 1×10⁷, 5×10⁷, 1×10⁶,5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴, 1×10³, 5×10³, (and so forth) cellsper administration, or any ranges between any two of the numbers, endpoints inclusive. In other embodiments, cells can be administered tosuch an individual by relative numbers of cells, e.g., said individualcan be administered about 1000 cells to up to about 10 billion cells perkilogram of the individual, such as at about, at least about, or at mostabout, 1×10⁸, 1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴,1×10³, 5×10³ (and so forth) cells per kilogram of the individual, or anyranges between any two of the numbers, end points inclusive. In someembodiments, between about 1 billion and about 3 billion cells areadministered to a patient. In other embodiments, the total dose may becalculated based on m² of body surface area, including 11×10¹¹, 1×10¹⁰,1×10⁹, 1×10⁸, 1×10⁷, per m². The average person is 1.6-1.8 m².

In one embodiment, the pathogenic infection is reduced by about 5%,about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about98%, about 99%, or 100% as compared to baseline or a control groupreceiving a conventional therapy.

In one embodiment, the pathogenic infection is reduced by about 5%,about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about98%, about 99%, or 100% as compared to baseline or a control groupreceiving a conventional therapy within about 1 day, about 2 days, about3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months,about 4 months, about 6 months, about 1 year after administering a firstdose of the genetically engineered microbes.

In one embodiment, the subject who is administered the geneticallyengineered microbes experiences and/or reports fewer or less severe sideeffects on natural gut microbiota as compared to baseline or a controlgroup receiving a conventional therapy. Any method known in the art forassessing natural gut microbiota can be used. Suitable methods include ahydrogen breath test or analyzing a bacterial culture of the smallintestine or a stool sample.

In one embodiment, the subject administered the genetically engineeredmicrobe experiences and/or reports fewer or less severe symptoms ascompared to baseline or a control group receiving a conventionaltherapy. Non-limiting examples of symptoms include, fever, pain inabdomen, fatigue, loss of appetite, headache, dry tongue, rice waterdiarrhea, diarrhea, severe diarrhea with vomiting, slow pulse, cold andclammy skin, dry tongue, severe dehydration, low blood pressure, loss ofweight, persistent cough, weakness, occasional blood in sputum, chestpain, burning in urine, painful urination, discharge of pus, yellow orgreen sputum, pain in chest while coughing, rapid pulse, excessiveperspiration, running nose, throat pain, sneezing, itching and burningin nose and eyes, vomiting, muscle pain, rash, itching, or anycombination thereof.

Non-limiting examples of conventional therapies for a pathogenicinfection include antibacterial drugs (e.g., penicillins,cephalosporins, macrolides, and fluoroquinolones), antiviral drugs(e.g., amantadine, rimantadine, oseltamivir, and zanamivir), andanti-fungal medication (e.g., clotrimazole, econazole nitrate,miconazole, terbinafine, fluconazole, ketoconazole, amphotericin). Insome embodiments, the conventional therapy is erythromycin, clindamycin,rifamycin, or any combination thereof.

The composition can be administered by any appropriate route, which willbe apparent to the skilled person depending on the disease or conditionto be treated. Typical routes of administration include oral, topical,intravenous, intra-arterial, intramuscular, subcutaneous, intracranial,intranasal or intraperitoneal. The composition can be administered byanother (e.g., a physician or healthcare provider) or self-administered.In one embodiment, the composition can be administered topically. Inanother embodiment, the composition can be administered via injection.In a preferred embodiment, the composition can be administrated orally.The terms “oral”, “enteral”, “enterally”, “orally”, “non-parenteral”,“non-parenterally”, and the like, refer to administration of a cell orcomposition to an individual by a route or mode along the alimentarycanal. Examples of “oral” routes of administration of a microbecomposition include, without limitation, swallowing liquid or solidforms of a microbe composition from the mouth, administration of amicrobe composition through a nasojejunal or gastrostomy tube,intraduodenal administration of a microbe composition, and rectaladministration, e.g., using suppositories that release a live microbedescribed herein to the lower intestinal tract of the alimentary canal.

The composition may also be directly delivered in any manner directlyinto the gastrointestinal tract. In one embodiment, it is delivereddownstream of the stomach directly into the intestines. In anotherinclude: (1) placement of a percutaneous endoscopic gastrostomy (PEG)tube and passing a weighted or non-weighted feeding tube into theduodenum or jejunum; (2) surgically placing a direct jejunostomy tube;or, (3) placing a tube directly into the jejunum with a known PEG-likeprocedure, whereby the jejunum is accessed by stab-piercing the jejunumfrom outside the abdominal wall.

The dosage and frequency of administration may depend on the type offormulation, route of administration, disease being treated, the amountof cells and any combination therapies, the subject's age, weight,gender, species, other conditions, and the like.

In some embodiments, the composition comprising genetically engineeredmicrobes is administered sequentially or concurrently with anantibiotic. In some embodiments, the genetically engineered microbes areadministered after a period of time following administration of anantibiotic. In some embodiments, the subject stops taking the antibioticprior to administration of the microbes.

EXAMPLES Example 1: Pathogen-Detecting Sensor Technology

A sensor technology that can turn any specific antibody or bindingdomain into a cell surface signal will be developed. The antibody orbinding domains will be fused to existing cell-surface receptor proteins(i.e., two-component signaling receptors, chemotaxis receptors, quorumsensing circuits) and the fusion proteins will be expressed on thesurface of the microbe. FIG. 1. The binding of a pathogen-specificligand to the antibody or binding domain will either (1) block ordisable the binding site of the original receptor, turning the ligandbinding into an OFF switch for the chosen signaling circuit or (2)induce a conformational change in the receptor to turn binding into anON switch. The binding event will then transduce a signal to drivespecific transcriptional responses and subsequent expression of specificeffectors, for example, a bacteriocin or a protease. Orthogonal bindingdomains will be designed for specific toxins and surface proteins.Targets include toxins TcdA and TcdB, which are secreted by the virulentform of C. difficile, and spore coat proteins (i.e., BclA glycoproteinsand cysteine-rich CdeC). Tying such a sensor to a chemotaxis pathwaywill allow the engineered microbes to “swim” toward virulent pathogens.

In addition, an infectious pathogen's own sensors can be targeted. Here,a pathogen genome will be mined for sensors of its own environmentallysecreted molecules (e.g., quorum sensing receptors). For example,microbes can be engineered to intercept C. difficile siderophores, ironscavenging molecules, to sense the presence of the pathogen and, at thesame time, deprive the pathogen of an essential resource.

Example 2: Targeted Countermeasures Against Virulent Pathogen Cells

Microbes will be engineered to produce countermeasures to kill, disableor modify the behavior of the virulent pathogen. Multiple proteins havebeen identified during a computational screen for native autolysins andC. difficile phage endolysins that may be able to specifically degradethe cell wall of virulent C. difficile to lyse the pathogen. As some ofthese lysin proteins are native proteins that play an essential role incell division, it would be difficult for the pathogen to evolveresistance to them. Proteins identified by the screen include thoseencoded by SEQ ID NOS. 1-40.

Candidate lysin proteins will be screened by high-throughput, cell-freesynthesis on a protein array to identify those that show cell-killingactivity against C. difficile but not the engineered microbes of thedisclosure. Antimicrobial peptides can also be screened for specificactivity in killing C. difficile. Effective lysins/peptides can beexpressed and secreted upon sensing the presence of the target pathogen.Alternatively, effective lysins/peptides can be produced in anencapsulated form in order to prevent potential toxicity to the hosttherapeutic agent. To release the encapsulated proteins, the engineeredmicrobe will turn on expression of a bacteriophage lytic protein uponpathogen detection. In addition to the lysins/peptides, encapsulatedantitoxins can also be releases to mitigate the effects of the virulenttoxins, for example, upon host tissues. The encapsulated shells can bemodified with antibodies for targeting to epithelial cells.

Microbes can also be engineered to deliver toxin-specific CRISPR guideRNA (gRNA) to disarm virulent C. difficile. Upon detection of virulentC. difficile, the prophage will be triggered to enter the lytic cycle,cause cell lysis of the engineered microbes and release the phage. Thephage can subsequently deliver the CRISPR gRNA to the virulent cell andeffectively edit the virulent cell genome to disable the toxin tcdA/Bgenes, converting the bacterium to a non-virulent form. The presence ofnon-toxin producing C. difficile can inhibit growth of the virulentform. Nagaro et al., (2013) Antimicrob. Agents Chemother.57(11):5266-5270. Therefore, by producing more non-virulent C.difficile, it is contemplated that suppression of the virulent form willbe stimulated.

Example 3: Targeted Countermeasures Against the Spore State

As a first strategy to selectively kill the highly resistant but dormantC. difficile spores, specific proteases that cleave spore coat proteinswill be used. An example of one such target is the cysteine-rich proteinCdeC, which has been shown to be essential for assembly of theexosporium layer. Paredes-Sabja et al., (2014) Trends Microbiol.22(7):406-416. Encapsulated proteases can be delivered, via host microbelysis, in which the encapsulin shell is targeted to CdeC to providespecificity of cleavage. As a second strategy, microbes can beengineered to metabolize primary bile salts into deoxycholate, which hasbeen shown to induce germination in C. difficile and also inhibit growthof the cells after germination. Sorg et al., (2008) J. Bacteriol. 190(7):2505-2512. By activating “sleeper” spores, the spores become visibleand vulnerable to the arsenal of agents the engineered microbes use totarget the virulent state of the pathogen.

Example 4: Engineered Microbes to Dynamically Adapt and Respond to thePathogen and Host State

Each countermeasure/agent produced by the engineered microbes to targetthe pathogen can be deployed under the control of an appropriate sensor.For example, the secretion of CRISPR-loaded phages to knockout thepathogen toxin genes can be under control of a toxin sensor, while thesecretion of spore coat degrading proteases can be under the control ofa spore coat sensor. Each of the feedback loops can be engineeredindependently (FIG. 2) and in parallel into multiple initial strains ofthe genetically engineered microbes and dose-response characteristicscan be fine-tuned through the choice of suitable transcription factors(TFs), multiplicity of TF binding sites, and autoregulation. Ang et al.,(2013) ACS Synth Biol. 2:547-567. It is preferred that responses thatinvolve cell lysis of the engineered microbe be have an all-or-nothingresponse, so only a fraction of the cells commit suicide. Eldar et al.(2010) Nature 467:167-173. Once each sensor-countermeasure system hasbeen fine-tuned individually, systems can be combined into a singleengineered microbe, with some additional regulatory logic to integrateacross sensor inputs and response pathways. FIG. 3 and FIG. 4

FIG. 5 shows an example of a dynamically adaptive therapeutic microbeaccording to an embodiment of the present disclosure that senses both C.difficile spores and virulent cells (i.e., contains a virulent sensorlinked to a virulent secretion system and a spore sensor linked to sporesection system). The therapeutic microbe/agent has been engineered todetect spore coat proteins. Binding of the spore coat protein to thesensor on the therapeutic agent triggers: (1) signal transductions tocause the therapeutic agent to secrete a compound that induces spores togerminate into a virulent form, for example, a bile salt or (2) releaseof encapsulated proteases that bind to and kill the C. difficile spores.The therapeutic agent also detects toxins released by the virulentcells. The toxins bind to the virulent sensor on the cell surface of theengineered microbe and activate expression of virulent agents. Forexample, the engineered microbe can release phages carrying CRISPR gRNAfor knockout of toxins whereby the phage infects the C. difficilepathogen and the native CRISPR system disrupts the toxin production toresult in a benign C. difficile. In addition, the engineered microbe canrelease encapsulated drugs, for example lysins. The lysins are releasedat low pH and kill the pathogenic C. difficile. Finally, the engineeredmicrobe can release encapsulated antitoxins to mitigate the toxin effecton host tissue (e.g., intestinal epithelium cells).

Example 5: Validation of Engineered Microbes

In vitro tests involving a simple two-bacterial system with theengineered microbe and C. difficile, both virulent and spore forms, willbe performed. Microscopy and flow cytometry will be utilized to examinehow effective the engineered microbes are in neutralizing both forms ofC. difficile. Next, combining the engineered microbes in a controlledmicrobiome, including non-virulent C. difficile and other gut microbes,the changes in the microcosm composition will be measured usingnext-generation sequencing.

Animal cell lines that are commonly used to model human intestinaldisease, for example Caco-2 and HT-29, will be introduced to theengineered microbes to assess the toxicity and efficacy of the microbesin the presence of host cells. Using in vivo animal studies, theengineered microbes will be administered, with and without other gutmicrobes, after an antibiotic treatment course in mice infected byvirulent C. difficile. The health state of the mice will be monitoredand the gut microbiome will be analyzed from the fecal microbiota usingnext generation sequencing.

Finally, in silico computer modeling will be employed to simulate thepopulation dynamics of the microbiome. Data from experimental in vitroand in vivo studies will be incorporated. A conceptual model will beformulated for the native behavior of the gut microbiome duringrecolonization after broad spectrum antibiotic treatment, with andwithout the presence of C. difficile, and the tipping point for theonset of C. difficile infection will be identified. Influence of theengineered microbes will then be incorporated into the system. Usingthese models, the impact of changing given factors (e.g., inoculum sizeand death rate) to the system will be predicted in order to assess theeffectiveness of the engineered microbes and predict the evolutionarybehavior of the gut microbiome components of interest.

What is claimed is:
 1. A genetically engineered viable Lactobacillus orLactococcus probiotic bacterium comprising a dual pathogen statedetection system, the dual pathogen detection system comprising: a firstsensor for detection of a large clostridial toxin (LCT) secreted by avirulent form of Clostridium difficile (C. diff), wherein the firstsensor is operatively connected to a virulent secretion system, whereinthe virulent secretion system produces one or more agents in anencapsulated shell and a second sensor for detection of a cell surfaceprotein on a spore form of C. diff, wherein the second sensor isoperatively connected to a spore secretion system, wherein the sporesecretion system produces an encapsulated agent.
 2. The geneticallyengineered probiotic bacterium of claim 1, wherein the first sensor isan antibody or a functionally active epitope binding fragment thereofthat immunospecifically binds to the large clostridial toxin and thesecond sensor is an antibody or an epitope binding fragment thereof thatimmunospecifically binds to the cell surface protein.
 3. The geneticallyengineered probiotic bacterium of claim 2, wherein the antibody is amonoclonal antibody or a polyclonal antibody.
 4. The geneticallyengineered probiotic bacterium of claim 1, wherein the large clostridialtoxin is C. difficile toxin A (TcdA) or C. difficile toxin B (TcdB). 5.The genetically engineered probiotic bacterium of claim 1, wherein thecell surface protein is a spore coat protein of C. difficile.
 6. Thegenetically engineered probiotic bacterium of claim 5, wherein the sporecoat protein is the BclA glycoprotein or the cysteine-rich protein CdeC.7. The genetically engineered probiotic bacterium of claim 1, whereinthe one or more agents in the encapsulated shell is an endolysin.
 8. Thegenetically engineered probiotic bacterium of claim 1, wherein the oneor more agents in the encapsulated shell is a bacteriocin selected fromthe group consisting of colicin, diffocin, pyocin, and rhuricin
 17. 9.The genetically engineered probiotic bacterium of claim 1, wherein theencapsulated agent induces germination of the spore.
 10. The geneticallyengineered probiotic bacterium of claim 1, wherein the encapsulatedagent is a bile salt or a protease.